Phorbol 12-myristate 13-acetate (PMA) affects a variety of cell processes, including proliferation and differentiation. In most normal cell types, PMA enhances proliferation by a protein kinase C (PKC)- dependent mechanism, while the proliferation of cancer cell lines is inhibited by PMA. PMA has been shown to induce cyclooxygenase-2 (COX-2) production in many cell lines. We examined the effect of PMA on functions of induced COX-2 on proliferation of head-and-neck UMSCC-22B cancer cells. PMA was found to inhibit head-and-neck cancer cell proliferation in a cell viability assay and to induce apoptosis. Abundance of p21 protein and p53 in these cells was increased by PMA treatment. The phorbol ester increased p21 expression in 22B cells through the classical PKC pathway and independently of activation of p53. In addition, PMA increased expression of COX-2 in a time-dependent, PKC-requiring manner. COX-2 siRNA increased abundance of both p53 and p21 in 22B cells and importantly potentiated the anti-proliferative effect of PMA. Confocal microscopy revealed that PMA increased nuclear accumulation of COX-2, but not COX-1, and that COX-2 formed complexes with p53, p21 and the coactivator, p300. Chromatin immunoprecipitation showed that nuclear fraction COX-2 and p53 bound to the promoter region of p21. PMA did not significantly increase PGE2 levels in cultured medium of 22B cells, but did increase by 13-fold the levels of PGE2 in the medium of immortalized normal (293T) cells. In conclusion, our results show that PMA inhibits cell proliferation in head-and-neck cancer cells by a mechanism that is potentiated by COX-2 siRNA. The latter results in an increase of p53 and p21 in PMA-treated cancer cells. In the absence of its siRNA, COX-2 is increased in PMAexposed cancer cells and becomes a nucleoprotein, where it complexes with p53 and p21. Thus, the antiproliferative activity of PMA in 22B cells is moderated by concurrent induction by PMA of COX-2 accumulation; this moderation may be due to reduction by COX-2 of availability of p21 and p53 in the nucleus.